Volume 22, Issue 7, Pages 1681-1694 (February 2018) Nuclear Organization in the Spinal Cord Depends on Motor Neuron Lamination Orchestrated by Catenin and Afadin Function  Carola Dewitz, Sofia Pimpinella, Patrick Hackel, Altuna Akalin, Thomas M. Jessell, Niccolò Zampieri  Cell Reports  Volume 22, Issue 7, Pages 1681-1694 (February 2018) DOI: 10.1016/j.celrep.2018.01.059 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Three-Dimensional Analysis of Motor Neuron Positions in the Developing Spinal Cord (A) Motor neuron organization in an E13.5 control embryo at lumbar spinal level. Isl1/2+, LMCm neurons; Hb9+, LMCl neurons; Isl1/2+, Hb9+, MMC neurons. (B) Digitally reconstructed distribution of LMC neurons at L1–L3, shown as a transverse projection. (C) Transverse contour density plots of LMCm (green) and LMCl (red) neurons. (D) Medio-lateral and dorso-ventral positions (mean position ± SD) of LMCm (green) and LMCl (red) neurons (#1, ○; #2, □; #3, ▵). (E and F) Boxplots showing distributions of LMCm (green) and LMCl (red) neurons along medio-lateral (E) and dorso-ventral (F) axes. (G) Correlation analysis of LMC positional coordinates. The scale bar indicates correlation values. For additional data examining LMC rostro-caudal positions, see Figure S1. Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Catenin Inactivation Perturbs Medio-lateral and Dorso-ventral Motor Neuron Organization (A and B) Organization of Isl1/2+ medial and Hb9+ lateral LMC neurons at lumbar spinal levels in E13.5 control (A) and βγΔMN (B) embryos. (C and D) Transverse contour density plots of LMCm (green) and LMCl (red) neurons in control (C) and βγΔMN (D) embryos. (E and F) Longitudinal contour density plots of LMCm (green) and LMCl (red) neurons in control (E) and βγΔMN (F) embryos. (G and H) Medio-lateral density plots of LMCm (green) and LMCl (red) neurons in control (G) and βγΔMN (H) embryos. (I) Average medio-lateral position of LMCm (green) and LMCl (red) neurons in control and βγΔMN embryos (mean ± SD; differences significant for LMCl neurons; t test, p < 0.001). (J) Average dorso-ventral position of LMCm (green) and LMCl (red) neurons in control and βγΔMN embryos (mean ± SD; differences significant for LMCm and LMCl neurons; t test: LMCm, p < 0.01; LMCl, p < 0.001). (K and L) Dorso-ventral density plots of LMCm (green) and LMCl (red) neurons in control (K) and βγΔMN (L) embryos. (M) Correlation analysis of LMC neuron positional coordinates in control and βγΔMN embryos. The scale bar indicates correlation values. (N) Average medio-lateral and dorso-ventral positions of LMCm (green) and LMCl (red) neurons in control and βγΔMN embryos (mean). For additional data regarding LMC organization in βγΔMN embryos, see Figure S2. Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Catenin Inactivation Disrupts Medio-lateral and Dorso-ventral Pool Segregation (A and B) Organization of H (Nkx6.1+), R/T (Nkx6.2+), and V (Er81+) motor pools in E13.5 control (A) and βγΔMN (B) embryos. The motor neuron area is delimited by a dashed line. (C and D) Transverse contour density plots of H (green), R/T (red), and V (blue) motor pools in control (C) and βγΔMN (D) embryos. (E and F) Longitudinal contour density plots of H (green, medial) and R/T (red, lateral) neurons in control (E) and βγΔMN (F) embryos. (G and H) Medio-lateral density plots of H (green, medial) and R/T (red, lateral) neurons in control (G) and βγΔMN (H) embryos. (I) Average medio-lateral position of H (green, medial) and R/T (red, lateral) neurons in control and βγΔMN embryos (mean ± SD; differences significant for H neurons; t test, p < 0.01). (J and K) Dorso-ventral density plots of H (green, ventral), R/T (red, ventral), and V (blue, dorsal) neurons in control (J) and βγΔMN (K) embryos. (L) Average dorso-ventral position of H (green), R/T (red), and V (blue) neurons in control and βγΔMN embryos (mean ± SD; differences significant for H, R/T, and V neurons; t test: H, p < 0.05; R/T and V, p < 0.001). For additional data regarding pool organization in βγΔMN embryos, see Figure S2. Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 N-Cadherin Elimination Does Not Interfere with Dorso-ventral Pool Segregation (A) Organization of Isl1/2+ medial and Hb9+ lateral LMC neurons at lumbar spinal levels in E13.5 NΔMN embryos. (B and C) Transverse (B) and longitudinal (C) contour density plots of LMCm (green) and LMCl (red) neurons in NΔMN embryos. (D) Medio-lateral density plots of LMCm (green) and LMCl (red) neurons in control (solid line), NΔMN (dashed line), and βγΔMN (dotted line) embryos. (E) Dorso-ventral density plots of LMCm (green) and LMCl (red) neurons in control (solid line), NΔMN (dashed line), and βγΔMN (dotted line) embryos. (F) Organization of H (Nkx6.1+), R/T (Nkx6.2+), and V (Er81+) motor pools in NΔMN embryos. The motor neuron area is delimited by a dashed line. (G) Transverse contour density plots of H (green), R/T (red), and V (blue) motor pools in NΔMN embryos. (H) Average distance between dorso-ventral positions of V-R/T (■) and V-H (▲) pools in control, NΔMN, and βγΔMN embryos (mean ± SD; differences significant for V-R/T p < 0.001: control versus βγΔMN, p < 0.001; NΔMN versus βγΔMN, p < 0.01; for V-H p < 0.01: control versus βγΔMN p < 0.01; one-way ANOVA and post hoc Tukey’s honest significant difference [HSD] test). (I) Dorso-ventral density plots of H (green, ventral), R/T (red, ventral), and V (blue, dorsal) neurons in control, NΔMN, and βγΔMN embryos. (J) Average dorso-ventral position of H (green, ventral), R/T (red, ventral), and V (blue, dorsal) neurons in control, NΔMN, and βγΔMN embryos (mean ± SD; differences significant for V neurons p < 0.001: control versus NΔMN and βγΔMN p < 0.001; for R/T neurons p < 0.001: control versus NΔMN and βγΔMN p < 0.001; for H neurons p < 0.01: control versus NΔMN and βγΔMN p < 0.01; one-way ANOVA and post hoc Tukey’s HSD test). For additional data regarding motor neuron organization in NΔMN embryos, see Figure S3. Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 Afadin Expression and Motor Neuron Generation in Developing Spinal Cord (A and B) Afadin mRNA (A) and protein (B) expression in E13.5 lumbar spinal cord. (C–F) Afadin expression in E13.5 lumbar spinal cord in control (C and D) and afadinΔMN (E and F) embryos. FoxP1 identifies LMC neurons. (G and H) Segregation of Lhx3+ MMC and FoxP1+ LMC neurons in E13.5 lumbar spinal cord of control (G) and afadinΔMN (H) embryos. (I) Number of MMC and LMC neurons found in E13.5 lumbar spinal cord of control (C) and afadinΔMN (●) embryos. Motor neurons/100 μm, mean ± SD. (J and K) pSMAD+, nNOS+ preganglionic column (PGC) neurons in E13.5 thoracic spinal cord of control (J) and afadinΔMN (K) embryos. (L) Number of HMC and PGC neurons in E13.5 thoracic spinal cord of control (C) and afadinΔMN (●) embryos. Motor neurons/100 μm, mean ± SD. For additional data regarding afadin and nectin expression in developing spinal cord, see Figure S4. Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 Perturbed Divisional and Pool Organization in Afadin Mutants (A–D) Isl1/2+ medial and Hb9+ lateral LMC neurons at L2/L3 in E13.5 control (A and C) and afadinΔMN (B and D) embryos. (E) Number of LMCm and LMCl neurons in E13.5 lumbar spinal cord of control (C) and afadinΔMN (●) embryos. Motor neurons/100 μm, mean ± SD. (F–I) Motor pools at L2/L3 in E13.5 control (F and H) and afadinΔMN (G and I) embryos. Nkx6.1+, Er81+ adductor/gracilis (A/G) neurons; Er81+, Nkx6.1− V neurons; Nkx6.2+ R/T neurons; Nkx6.1+, Er81− H neurons. (J) Number of Er81+, Nkx6.1+, and Nkx6.2+ motor neurons in E13.5 lumbar spinal cord of control (C) and afadinΔMN (●) embryos. Motor neurons/100 μm, mean ± SD. Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 Motor Pool Positioning Defects in Afadin Mutants (A and B) Transverse (A) and longitudinal (B) contour density plots of LMCm (green) and LMCl (red) neurons in E13.5 afadinΔMN embryos. (C) Boxplots showing the distribution of LMCm (green) and LMCl (red) neurons along the medio-lateral axis in control and afadinΔMN embryos. (D) Average medio-lateral position of LMCm (green) and LMCl (red) neurons in control and afadinΔMN embryos (mean ± SD; differences significant for LMCl neurons; t test, p < 0.01). (E and F) Transverse (E) and longitudinal (F) contour density plots of H (green, medial) and R/T (red, lateral) motor neurons in E13.5 afadinΔMN embryos. (G) Boxplots showing the distribution of H (green) and R/T (red) neurons along the medio-lateral axis in control and afadinΔMN embryos. (H) Transverse contour density plots of H (green) motor neuron and LMCm (gray) neuron areas in afadinΔMN and control embryos, respectively. (I and J) Transverse contour plots of R/T (red, ventral) and V (blue, dorsal) motor neurons (I) and H (green, ventral) and V (blue, dorsal) neurons (J) in control and afadinΔMN embryos. (K) Box-plots showing distributions of H (green, ventral), R/T (red, ventral), and V (blue, dorsal) neurons on the dorso-ventral axis in control and afadinΔMN embryos. (L) Average dorso-ventral position of H (green), R/T (red), and V (blue) neurons in control and afadinΔMN embryos (mean ± SD). (M) Average distance between dorso-ventral positions of V-R/T (■) and V-H (▲) pools in control and afadinΔMN embryos (mean ± SD). For additional data regarding motor neuron organization and cadherin/catenin expression and function in afadinΔMN embryos, see Figures S5 and S6. Cell Reports 2018 22, 1681-1694DOI: (10.1016/j.celrep.2018.01.059) Copyright © 2018 The Author(s) Terms and Conditions